The domestic cat (<i>Felis catus</i>) is an established animal model for studies of the brain, genetics, pharmacology, and nutrition. In addition, the cat serves as a model for viral infectious diseases including Feline Immunodeficiency Virus (FIV) which shares features with Human Immunodeficiency Virus (HIV), Feline Leukemia Virus (FeLV) an oncogenic pathogen of cats and Feline Foamy Virus (FFV) has not been firmly linked to any disease. Although molecular and genetic features of these feline retroviruses have been unraveled, the contribution of host genes in permissiveness towards virus replication, determining disease, and influencing spread and transmission are only now becoming possible due to new achievements in genomics. Recently, the lightly covered (1.9X genome coverage) whole genome shotgun sequences of the domestic cat were assembled and annotated based on the comparison with conserved sequence blocks of the genome sequences of human and dog. Using this resource, we identified gene sequences orthologous to cytidine deaminases AICDA (AID) and APOBEC1, 2, and 4 in the domestic cat genome and two antiretroviral APOBEC3 sequences contained on seperate scaffolds: one nearly identical to feA3H (Genbank: EF173020.1) containing 5 exons with 797/798 nucleotide sequence identities and a second partial sequence related to the published cat feA3C cDNA (AY971954) containing 2 exons with 613/619 nucleotide sequence identities but missing the 5' 251 bp. To further characterize A3 genes in the Abyssinian cat genome, we studied fosmids that had been end-sequenced as part of the 1.9X domestic cat genome project from Agencourt Bioscience Corporation. We established a web-based fosmid cloning system, the 1806 fosmid 384-well plates were stored in assigned locations. A fosmid database of 1,288,606 fosmid clones, sequence-trace-IDs, plate and well IDs, and freezer location ID was generated and linked to GARFIELD browser and NCBI trace ID. In this system, fosmid cloning is achieved by using potential orthologues (i.e., human or mouse or dog or yeast) of genes of interest and search for fosmid trace IDs by (i) gene ID/symbol in GARFIELD browser or by (ii) discontinuous megablast of orthologous sequences to Felis catus WGS in NCBI blast site. With the trace ID, the fosmid freezer location ID can be retrieved from the fosmid database. We have tested 704 fosmids and could identify with 99% accuracy 616 of them (87.5%), as confirmed by fosmid end sequencing. Using this system, we selected a total of seven fosmids that encompassed the A3 genes and three clones were analyzed by nucleotide sequencing. Gene modeling demonstrated the presence of three feline A3C genes designated A3Ca (identical to feA3C cDNA), A3Cb and A3Cc and a single A3H gene, arrayed in a head-to-tail formation spanning 32 kb of the 50 kb region sequenced. The A3C genes each consist of four exons with coding sequences that spanned 3693, 6457 and 6498 bp for A3Cc, A3Ca, and A3Cb respectively, whereas A3H contained one 5untranslated exon (UTR) followed by four coding exons that spanned 2237 bp. Consensus splice donor sites were observed for exons 1-4 in A3H and in all four coding exons of the three A3C genes. Interestingly, splicing at the splice donor sites of exon 4 in all A3C genes eliminates the overlapping termination codon of the fe3 cDNA (CTT AGG TGA) potentially allowing the generation of chimeric read-through transcripts. We used diagnostic PCR primers to analyze RNA expression of these genes. In MYA-1 cells A3Ca, -b and c genes are expressed but CrFK and KE-R cells expressed only A3Ca. Feline A3H was detected in all analyzed cell lines and PBMCs. As expected, we also detected a cDNA composed of the fused open reading frames of A3C and -3H, designated A3CH. A closer inspection of the sequence revealed that the transcript is encoded by exons 1 - 3 of A3Ca, exon 4 of A3Cb and exons 2 - 5 of A3H. The double-domain A3CH RNA was found in 3 tested cell lines (CrFK, MYA-1, KE-R) and feline PBMCs. Using reporter systems for feline retroviruses, we found that specific feline A3 proteins selectively inactivate only defined genera of feline retroviruses: Bet-deficient FFV was mainly inactivated by feA3Ca, -b, c, while feA3H and feA3CH were only weakly active. In contrast, the infectivity of Vif-deficient FIV and FeLV was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. The coding sequences of the genes A3Ca, A3Cb and A3Cc show 97.6 98.9% identical nucleotides and 96.3 96.5% identical amino acids to each other. The predicted proteins of A3Cb and A3Cc differ in six or seven amino acids from A3Ca. The feline A3H shows highest similarity to the human A3H and dog A3H, with 44.4% and 46.3% identical amino acids respectively. The feline A3C genes show high overall similarity to human A3C with 43.3 43.8% identical amino acids. In addition to the high degree of sequence identity between the coding sequences of the three feline A3C genes, the pattern of repetitive elements and the pairwise percent identities between the introns of the three genes: the introns of A3Ca and A3Cb have a high degree of nucleotide sequence identity (98-99%) across all three introns whereas A3Cc showed a lower degree of sequence identity to either A3Ca or A3Cb (67-96%) depending on the size of the intron. Phylogenetic analyses indicate that this gene triplication occurred likely by two consecutive duplication steps: one ancestral A3C gene duplicated to the ancestor of A3Cc and a second gene, which later duplicated giving rise to A3Ca and A3Cb genes. Assuming that the average genomic mutation rate is 0.1% per million years, we can conclude that the A3Ca, A3Cb split occurred 8 13 million years ago suggesting that the final A3C gene duplication occurred at the time of radiation of Felidae. It is generally believed, that the evolution of new protein functions after gene duplication plays an important role in the evolution of the diversity of organisms and typically allows for an increased specialization or functional gain of the daughter genes. In light of the seven A3 genes in primates, it is tempting to speculate, that cats like primates were under a specific evolutionary pressure to amplify the diversity of the co-expressed A3 proteins that provided additional fitness. Other mammals, like rodents and dogs were either not faced by these infectious agents or managed to counteract retroviruses and related retroid elements it in a way not involving A3